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Biology 2290F/G
Irene Krajnyk

Bio 2290G Winter/2013 Zabulionis Unit Rm 330 NCB ROTATIONS 1-4 Session 1: Welcome: Transformation of E. coli Welcome to the first lab session of the Zabulionis Unit of Biology 2290F—Scientific Methods in Biology. Beginnings are very important; during this session we’ll get to know one another, and have a look at what is in store for the 6 sessions in this unit. It is much better to be proactive than reactive — read, understand, and be prepared for each session. In the laboratory sessions following the first two, you will design and carry out experiments to answer a question of your choice regarding the biology of bacteria and plasmids. Some of the biological context for your experiments will be presented today. This laboratory session also introduces some conceptual background and technical skills through exercises involving transformation of E. coli bacteria. Objectives: Course and Zab Unit structure. Explain the steps involved in the transformation of E. coli. Unit resources. Micro-pipet usage (please don’t wreck them!!!) Safety: fire extinguisher, shower, gas shut- Set up Lab book according to guidelines in the off, eye wash station, EMERGENCY. Resource Manual (pg 90 - 91) and Unit style. Transform E. coli cells with plasmid DNA. Basic Transformation Flow Chart: E.coli JM101 cells on pick plate ↓ add to cold calcium chloride ↓ ice ↓ divide into 2 tubes ↓ ↓ add TE add plasmid ↓ ↓ ice ↓ ↓ 45 C heat pulse ↓ ↓ ice ↓ ↓ add LB broth ↓ ↓ 0 37 C incubation ↓ ↓ plate ↓ ↓ incubate When recording a protocol in your lab book—can someone pick up your lab book and do the experiment exactly the way you did it??? Micropipette Usage: The Gilson (navy blue) pipettors are the main ones we use; all of them have 3 digits in their display but each means a different volume. When selecting a pipettor, make sure its pipetting range includes the volume you want to pipet; i.e. if you want to pipet 500 µL, do not use the p200 pipettor since its range is 50 µL to 200 µL. Name of pipettor: Range: Colour of tip: Volume example: P1000 - blue cap 200 µL to 1000 µL blue 050 = 500 µL P200 - yellow cap 50 µL to 200 µL yellow 050 = 50 µL P20 - yellow cap 1.0 µL to 20.0 µL yellow 050 = 5.0 µL We also have Eppendorf pipettors (cream coloured) which have a range of 10.0 µL to 100.0 µL; e.g. 0500 = 50.0 µL and they take a yellow tip (don’t forget the decimal). A number of these pipettes have already been destroyed last term—please, be very careful with these; ask if you are in the least bit unsure about the number or how to change them. Do not try to dial below or above the range of the pipettor you are using—you are destroying the pipettor if you do. None of the pipettors should make any noise when changing the volume on the dial. Learn what the numbers mean on the different pipettors. Quiz #1 will include a question about the pipettors and pipetting. Safety: Lab coats, long pants, and glasses required for bench work. NO open-toed shoes. Long- hair tied back. NO food or drink in the lab. Beware of hazards associated with Bunsen burners, handling of bacteria etc.. BE AWARE OF WHAT YOU ARE DOING!!! Background Prep: Be aware of what the numbers mean and the ranges of the micropipettors mentioned above. Read the “Practical” file on OWL so you are aware of the skills we will be testing you in Session 6 and you will be learning in this lab period. Read the following: Antibiotics (ampicillin and kanamycin), Aseptic technique (Pouring plates, Transfer techniques, Pipetting tips), Micropipettes, Plasmids, Safety in the Laboratory, Scientific Nomenclature, and Transformation background material in Resource Manual. Assignment(s): “Exit Stamp” for Transformation Protocol in Lab Book. Session 2: Transformation Background The first experiment will provide context for a discussion of basic principles underlying transformation and antibiotic resistance in bacteria. The relevance and importance of controls will also be discussed along with proper data recording procedures. Most of session 3 is devoted to designing experiments. We will begin that process in this class by introducing the “Toy Box”. Objectives: Explain the relevance and importance of controls in Describe the mechanism of transformation and experimental designs. Discuss antibiotics and the structure of an R-plasmid resistance. What is a transformant? What is a satellite colony? Initiate your Independent Experiment by Develop a raw data table. discussing the “Toy Box” Background Prep: Review Antibiotics and Plasmids. Look at Independent Experimental Design (Antibiotic Physiology, Curing and Mutagenesis) in the resource manual and the Requisition Form on OWL. Session 3: Experimental Design We will begin with a short Quiz (2%) which may include questions of micropipette usage, the transformat
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